Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity.In nature, plants are constantly exposed to a large number of pathogens including fungi, bacteria, and viruses. In response, plants have evolved multiple layers of defense mechanisms to resist the pathogen attack (1). The first line of plant defense against fungi is penetration resistance that is achieved by localized cell wall appositions (CWAs), also called papillae, on an inner surface of cell walls at the site of fungal penetration (2). CWAs consist primarily of callose (β-1,3-glucan), lignin, cell wall proteins, and reactive oxygen species (2–4). The focal deposition of these elements in papillae appears to be an early and essential factor in plant penetration resistance (5).Studies on the genetic basis of penetration resistance have revealed that entry control of A. thaliana against nonadapted powdery mildews largely depends on several PENETRATION (PEN) genes (PEN1, PEN2, and PEN3). All three PEN proteins are also recruited to attempted fungal penetration sites (6–11). Intriguing findings show that the focal accumulation of PEN1 and PEN3 occurs outside the plasma membrane and within papillae or haustorial encasements (3, 11, 12). Disruption of actin cytoskeleton by pharmacological inhibitors blocks PEN3–GFP accumulation at most penetration sites, but has a lesser effect on the recruitment of GFP–PEN1 to these sites (11), suggesting that transport pathways mediating PEN1 and PEN3 recruitment and export to the apoplastic papillae are distinct.Accumulation of dynamically moving cytoplasm near the pathogen penetration site is the most striking and microscopically visible early response in epidermal cells (2). The secretory vesicles and organelles, including peroxisomes, Golgi, mitochondria, and the nucleus, also move toward penetration sites (7, 13). In addition to the deposition of cell wall reinforcements and focal accumulation of penetration-related proteins such as PEN3, the accretion of cytoplasm and organelles at sites of attempted fungal penetration involves reorganization of actin cytoskeleton, which forms a radial array focused on penetration site (10, 11, 14–18). Consistent with this finding, disruption of AFs hampers penetration resistance, leading to increased penetration frequency by various fungal and oomycete pathogens (15–17, 19). However, the mechanisms that drive AF dynamics and active transport of cellular components toward sites of attempted pathogen penetration remain elusive. Myosins are molecular motors responsible for AF-based motility (20). Recently, plant class XI myosins were implicated in the organization of actin cytoskeleton, organelle and vesicle transport, cell expansion, and plant growth (21–27). Although none of the individual myosin gene knockouts produces plant growth defects (22), progressive elimination of two to four highly expressed myosins results in concomitant reduction in cell and plant size (23, 24). However, relatively little is known about the functions of myosins in plant–pathogen interactions (28, 29).Using pharmacological and genetic approaches to disrupt myosin function in Arabidopsis, we show that transient assembly and polarization of actin filament (AF) bundles toward the fungal penetration site are regulated by myosin motors. Furthermore, we demonstrate that plant myosins contribute to focal aggregation of a battery of cellular defense activities at the infection site and papillary deposition of cell wall appositions of lignin-like compounds, callose and PEN1, and are required for plant penetration resistance.     

Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.The auxin binding protein 1 (ABP1) was first isolated from maize plants based on its ability to bind auxin (1). The crystal structure of ABP1 demonstrated clearly that ABP1 has an auxin-binding pocket and, indeed, binds auxin (2). However, the elucidation of the physiological functions of ABP1 has been challenging because the first reported abp1 T-DNA insertion mutant in Arabidopsis was not viable (3). Nevertheless, ABP1 has been recognized as an essential gene for plant development and as a key component in auxin signaling (4–9). Because viable abp1 null mutants in Arabidopsis were previously unavailable, alternative approaches have been used to disrupt ABP1 function in Arabidopsis to determine the physiological roles of the protein. Cellular immunization approaches were used to generate ABP1 knockdown plants (10, 11). Inducible overexpression of the single chain fragment variable regions (scFv12) of the anti-ABP1 monoclonal antibody mAb12 both in cell lines and in Arabidopsis plants presumably neutralizes the endogenous ABP1 activities (10, 11). Two such antibody lines, SS12S and SS12K, have been widely used in many ABP1-related studies (4, 6, 9–11). The results obtained from the characterization of the antibody lines suggest that ABP1 regulates cell division, cell expansion, meristem activities, and root development (4, 6, 10, 12, 13). Transgenic plants that overexpress ABP1 antisense RNA were also used to elucidate the physiological functions of ABP1 (4, 10). Moreover, missense point mutation alleles of abp1 have also been generated through the Arabidopsis TILLING project. One such TILLING mutant, named abp1-5, harbors a mutation (His94 >Tyr) in the auxin-binding pocket and has been widely used in many ABP1-related studies (4, 8, 9). Previous studies based on the antisense lines, antibody lines, and Arabidopsis mutant alleles have led to the conclusion that ABP1 is essential for embryogenesis, root development, and many other developmental processes. However, the interpretation of results generated by using the ABP1 antisense and antibody lines are not straightforward and off-target effects have not been completely ruled out. We believe that characterization of abp1 null plants is urgently needed to unambiguously define the roles of ABP1 in auxin signaling and in plant development.In the past several years, studies of the presumed ABP1-mediated auxin signal transduction pathway were carried out in several laboratories. It has been hypothesized that ABP1 is an auxin receptor mediating fast, nongenomic effects of auxin (4–6, 8, 9), whereas the TIR1 family of F-box protein/auxin receptors are responsible for auxin-mediated gene regulation (14, 15). One of the proposed functions of ABP1 is to regulate subcellular distribution of PIN auxin efflux carriers (6, 9, 13). Furthermore, a recent report suggests that a cell surface complex consisting of ABP1 and transmembrane receptor-like kinases functions as an auxin receptor at the plasma membrane by activating the Rho-like guanosine triphosphatases (GTPases) (ROPs) in an auxin-dependent manner (8). ROPs have been reported to play a role in regulating cytoskeleton organization and PIN protein endocytosis (5, 6). However, it is important to unequivocally determine the biological processes that require ABP1 before extensive efforts are directed toward elucidating any ABP1-mediated signaling pathways.In this paper, we generate and characterize new abp1 null mutants in Arabidopsis. We are interested in elucidating the molecular mechanisms by which auxin regulates flower development because our previously identified auxin biosynthetic mutants display dramatic floral defects (16–18). Because ABP1 was reported as an essential gene and ABP1 binds auxin (2, 3), we decided to determine whether ABP1 plays a role in flower development. We used our recently developed ribozyme-based CRISPR gene editing technology (19) to specifically inactivate ABP1 during flower development. Unexpectedly, we recovered a viable abp1 mutant (abp1-c1, c stands for alleles generated by using CRISPR) that contains a 5-bp deletion in the first exon of ABP1. We also isolated a T-DNA abp1 allele (abp1-TD1) that harbors a T-DNA insertion in the first exon of ABP1. We show that both abp1-c1 and abp1-TD1 are null mutants. Surprisingly, the mutants were indistinguishable from wild-type (WT) plants at all of the developmental stages we analyzed. Our data clearly demonstrate that ABP1 is not an essential gene and that ABP1 does not play a major role in auxin signaling and Arabidopsis development under normal growth conditions.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field

Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana. To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics of A. thaliana.The genomes of eukaryotes originate from ancient endosymbiotic associations that eventually led to energy-harnessing organelles: mitochondria, common to all eukaryotes, and chloroplasts in the “green” lineage. The evolution of endosymbionts into cellular organelles was accompanied by massive gene loss, with a large proportion being transferred to the nucleus (1, 2). Nevertheless, mitochondria and chloroplasts retained a few (30–80) protein-encoding genes that play crucial roles in energy metabolism (respiration and photosynthesis). Mitochondrion and chloroplast metabolisms rely on the proper interaction of nuclear-encoded proteins and their counterparts encoded in the organelle genome. Consequently, the genes in nuclear and organellar compartments are expected to be coadapted (3).Cytonuclear coadaptation has been demonstrated by altered phenotypes observed on interspecific exchanges of cytoplasm between related species in mammals (4), yeast (5), arthropods (6), and plants, whose interspecific crosses are frequently successful (7). These alterations affect organelle function and even the organism phenotype, indicating epistasis between nuclear and cytoplasmic genes. Although cytonuclear coadaptation is generally studied at the interspecific level, the existence of intraspecific genetic diversity in organelle genomes suggests a potential for genomic coadaptation within species. A few studies have reported phenotypic effects of intraspecific cytonuclear epistasis in nonplant species (8–11). In plants, many studies have focused on cytoplasmic male sterility (CMS), an impairment of pollen production governed by nucleo-mitochondrial interactions in some hermaphroditic species (12), in particular in crops and their relatives (13). The phenotypic effects of intraspecific cytonuclear epistasis other than CMS have been reported in only a limited number of plant systems (14–17), with evidence that cytoplasmic variation contributes to local adaptation (18, 19).In recent years, several studies using reciprocal segregating populations of the model plant Arabidopsis thaliana have investigated the effect of cytonuclear epistasis on a number of laboratory-measured phenotypes such as the metabolome, defense chemistry and growth (17, 20, 21), water-use efficiency (22, 23), and seed germination (24, 25). Although some studies have reported significant effects of cytonuclear epistasis (17, 20, 21, 23, 25), others have found additive cytoplasmic effects but with weak or no cytonuclear epistasis (22). Each of these studies (with the exception of ref. 25) was, however, based on a single reciprocal cross between two natural accessions, thereby preventing the estimation of the prevalence of cytonuclear epistasis in this species. In addition, although these reports involve adaptive traits (26–30), the investigation of the effect of cytonuclear epistasis on adaptive phenotypes in field conditions is, at best, scarce in A. thaliana.Here, following the modern standards of ecological genomics (31), we explored the prevalence of cytonuclear interactions on adaptive whole-organism traits in the model plant A. thaliana in a field experiment. To do so, based on eight natural accessions of a core collection that covers a significant part of the species’ cytoplasmic and nuclear genetic diversity in A. thaliana (25, 32), we created eight series of seven cytolines. Cytolines are genotypes that combine the nuclear genome from one parent with the organelle genomes of another (33). We examined the cytolines and their parental accessions for effects of cytonuclear interactions on 28 field-measured traits related to germination, phenology, resource acquisition, plant architecture and seed dispersal, fecundity, and survival.     

Identification of divergent type VI secretion effectors using a conserved chaperone domain

The type VI secretion system (T6SS) is a lethal weapon used by many bacteria to kill eukaryotic predators or prokaryotic competitors. Killing by the T6SS results from repetitive delivery of toxic effectors. Despite their importance in dictating bacterial fitness, systematic prediction of T6SS effectors remains challenging due to high effector diversity and the absence of a conserved signature sequence. Here, we report a class of T6SS effector chaperone (TEC) proteins that are required for effector delivery through binding to VgrG and effector proteins. The TEC proteins share a highly conserved domain (DUF4123) and are genetically encoded upstream of their cognate effector genes. Using the conserved TEC domain sequence, we identified a large family of TEC genes coupled to putative T6SS effectors in Gram-negative bacteria. We validated this approach by verifying a predicted effector TseC in Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the cognate immunity protein. Further, we demonstrate that TseC secretion requires its cognate TEC protein and an associated VgrG protein. Distinct from previous effector-dependent bioinformatic analyses, our approach using the conserved TEC domain will facilitate the discovery and functional characterization of new T6SS effectors in Gram-negative bacteria.Protein secretion systems play a pivotal role in bacterial interspecies interaction and virulence (1, 2). Of the known secretion systems in Gram-negative bacteria, the type VI secretion system (T6SS) enables bacteria to compete with both eukaryotic and prokaryotic species through delivery of toxic effectors (2–4). The T6SS is a multicomponent nanomachine analogous to the contractile bacteriophage tail (5). First characterized in Vibrio cholerae (6) and Pseudomonas aeruginosa (7), the T6SS has now been identified in ∼25% of Gram-negative bacteria, including many important pathogens (2, 8), and has been implicated as a critical factor in niche competition (9–11).The T6SS structure is composed of an Hcp inner tube, a VipAB outer sheath that wraps around the Hcp tube, a tip complex consisting of VgrG and PAAR proteins, and a membrane-bound baseplate (2, 4, 12). Sheath contraction drives the inner Hcp tube and the tip proteins, VgrG and PAAR, outward into the environment and neighboring cells (13, 14). The contracted sheath is then dissembled by an ATPase ClpV and recycled for another T6SS assembly and contraction event (12, 15, 16). Two essential T6SS baseplate components, VasF and VasK, are homologous to the DotU and IcmF proteins of the type IV secretion system (T4SS) in Legionella pneumophila (17).Bacteria often possess multiple copies of VgrG and PAAR genes that form the tip of T6SS, and deletion of VgrG and PAAR genes abolishes T6SS secretion (14). Some VgrG and PAAR proteins carry functional extension domains and thus act as secreted T6SS effectors, as exemplified by the VgrG1 actin cross-linking domain (6), VgrG3 lysozyme domain in V. cholerae (18, 19), and the nuclease domain of the PAAR protein RhsA in Dickeya dadantii (20). Known T6SS effectors can target a number of essential cellular components, including the actin and membrane of eukaryotic cells (18, 21, 22) and the cell wall, membrane, and DNA of bacterial cells (3, 18–20, 23, 24). Each antibacterial effector coexists with an antagonistic immunity protein that confers protection during T6SS-mediated attacks between sister cells (3, 18, 24). Interestingly, T6SS-mediated lethal attacks induce the generation of reactive oxygen species in the prey cells (25), similar to cells treated with antibiotics (26, 27).For non-VgrG/PAAR–related effectors, their translocation requires either binding to the inner tube Hcp proteins as chaperones or binding to the tip VgrG proteins (2, 14, 28). T6SS-dependent effectors can be experimentally identified by comparing the secretomes of WT and T6SS mutants (3, 29–31) and by screening for T6SS-encoded immunity proteins (18). Because known effectors lack a common secretion signal, bioinformatic identification of T6SS effectors is challenging. A heuristic approach based on the physical properties of effectors has been used to identify a superfamily of peptidoglycan-degrading effectors in bacteria (32). A recent study identified a common N-terminal motif in a number of T6SS effectors (31). However, this motif does not exist in the T6SS effector TseL in V. cholerae (18).In this study, we report that VC1417, the gene upstream of tseL, encodes a protein with a highly conserved domain, DUF4123. We show that VC1417 is required for TseL delivery and interacts with VgrG1 (VC1416) and TseL. Because of the genetic linkage of VC1417 and TseL and its importance for TseL secretion, we postulated that genes encoding the conserved DUF4123 domain proteins are generally located upstream of genes encoding putative T6SS effectors. Using the conserved domain sequence, we bioinformatically predicted a large family of effector proteins with diverse functions in Gram-negative bacteria. We validated our prediction by the identification and characterization of a new secreted effector TseC and its antagonistic immunity protein TsiC in Aeromonas hydrophila SSU. Our results demonstrate a new effective approach to identify T6SS effectors with highly divergent sequences.     

Natural variation of rice strigolactone biosynthesis is associated with the deletion of two MAX1 orthologs

Rice (Oryza sativa) cultivar Azucena—belonging to the Japonica subspecies—exudes high strigolactone (SL) levels and induces high germination of the root parasitic plant Striga hermonthica. Consistent with the fact that SLs also inhibit shoot branching, Azucena is a low-tillering variety. In contrast, Bala, an Indica cultivar, is a low-SL producer, stimulates less Striga germination, and is highly tillered. Using a Bala × Azucena F6 population, a major quantitative trait loci—qSLB1.1—for the exudation of SL, tillering, and induction of Striga germination was detected on chromosome 1. Sequence analysis of the corresponding locus revealed a rearrangement of a 51- to 59-kbp stretch between 28.9 and 29 Mbp in the Bala genome, resulting in the deletion of two cytochrome P450 genes—SLB1 and SLB2—with high homology to the Arabidopsis SL biosynthesis gene, MAX1. Both rice genes rescue the Arabidopsis max1-1 highly branched mutant phenotype and increase the production of the SL, ent-2′-epi-5-deoxystrigol, when overexpressed in Bala. Furthermore, analysis of this region in 367 cultivars of the publicly available Rice Diversity Panel population shows that the rearrangement at this locus is a recurrent natural trait associated with the Indica/Japonica divide in rice.The root parasitic Striga spp. parasitize on roots of crops in tropical and subtropical areas. The species typically parasitize cereals, including economically important crops such as maize, sorghum, millet, and rice (1). The parasitic relationship is dependent on the ability of the parasite to detect the host, which is mediated by the perception of strigolactones (SLs), molecules exuded by the host into the rhizosphere, by the seeds of the parasite (2). SLs are also signaling molecules for the establishment of the symbiosis with arbuscular mycorrhizal (AM) fungi (3) that help the plant to improve nutrient uptake. Under low phosphate availability, SL exudation into the rhizosphere is strongly enhanced, hence promoting AM symbiosis (4). As a negative consequence, however, agricultural areas with poor soils and low fertilizer input are strongly affected by Striga (1, 4). In addition to their rhizosphere role, SLs also function as plant hormones inhibiting shoot branching and modulating root architecture (5–7), also in response to phosphate deficiency (8, 9). SL biosynthesis or signaling mutants have increased axillary bud outgrowth, resulting in a bushy and dwarf phenotype (10). Biosynthesis of SLs proceeds through isomerization of β-carotene by β-CAROTENE ISOMERASE (D27), followed by cleavage by CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CAROTENOID CLEAVAGE DIOXIGENASE 8 (CCD8), which results in the formation of carlactone (11–16). The gene(s) responsible for the conversion of carlactone to a SL has/have not been identified, although MORE AXILLARY GROWTH 1 (MAX1), encoding a cytochrome P450 (CYP) in Arabidopsis, has been suggested to be a candidate (8, 11, 17, 18). SL signaling is mediated by an F-Box protein (MAX2 in Arabidopsis; D3 in rice) and an α/β-hydrolase protein (D14) (5, 6, 19, 20).In the present study, molecular genetics was used to further elucidate the SL biosynthetic pathway. We had observed that the rice cultivars Bala and Azucena differ greatly in SL biosynthesis and susceptibility to Striga infection. The Bala × Azucena F₆ recombinant inbred line (RIL) population was used to map quantitative trait loci (QTL) related to SLs. A major QTL was detected explaining most of the variation in the concentrations of all five SLs detected in rice exudates. This locus was also detected as a QTL for rice–Striga interaction in a previous study that used the same population (21). Here we show that the QTL is due to a rearrangement of a 51- to 59-kbp stretch between 28.9 and 29.0 Mbp of chromosome 1 in the Bala genome. This rearrangement results in the deletion of two CYP genes, which we show are orthologs of the Arabidopsis MAX1. The rearrangement of this locus is a recurrent natural trait, observed in several rice cultivars.